CN104531895A - Method for determining virus titer - Google Patents
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Abstract
The invention provides a method for determining virus titer. In the method, side scatter variation of virus-infected cells is utilized for determining the titer of virus. The method avoids the problems, generally existing in the prior art for determining virus titer, that a determination result is too strong in subjectivity and is not enough accurate, and the method is capable of objectively recording data, and the determination result of the method has superior accuracy and precision. The method also has the advantages of simple operation and relatively low cost, and is relatively widely applicable.
Description
Technical field
The invention belongs to detection analysis field, be specifically related to a kind of method measuring virus titer.
Background technology
In order to expression alien gene in insect or mammalian body, the mode of infectious viral particle host cells infected usually can be adopted to realize.That is, adopt the virus expression carrier inserting foreign gene, import in insect cell or mammalian cell, make exogenous gene expression.Common virus expression carrier has: baculovirus transfer vector, adenovirus expression carrier, retrovirus expression vector, Lentiviral etc.In raising exogenous gene expression amount, optimizing the suitableeest virus infection amount (MOI) is one of important means, therefore needs accurate quantification as the virus titer of expression vector.
Virus titer measuring method conventional at present comprises plaque ethods, Endpoint Dilution Method, euzymelinked immunosorbent assay (ELISA), colorimetric indicator method, fluorescence quantifying PCR method etc.Aforesaid method is specifically:
1) plaque ethods measures the most classical method of virus titer, and main operational steps is: to connect after poison at cell upper berth agar gel, cultivate 5-8 days, counting plaque, by plaque number and dilution conversion, obtain virus titer.But, plaque ethods complex operation, and require that operator has certain operating experience, there is certain restriction in its application.
2) main operational steps of Endpoint Dilution Method is: with 50% TCID (TCID
50) represent, by virus according to inoculating cell after doubling dilution or gradient dilution, cultivate 5-8 days, examine under a microscope cytopathy (CPE) situation, judge whether each hole infects.The method needs virus infected cell, and makes cell occur obvious CPE, more by rule of thumb judge whether infects, therefore, when cytopathy is not fairly obvious, have undetected possibility, namely subjectivity and the impact of sense datum on detected result larger.
3) euzymelinked immunosorbent assay (ELISA) utilizes the specific antibody for virus envelope protein, and direct labeled virus particle, to measure virus titer.The antibody that the method needs is usually expensive, and in addition, the virus utilizing this method to measure includes the pseudovirion of replication defective, causes titre overestimate.
4) colorimetric indicator method is by detecting cytoactive, thus indirectly judges whether each hole infects.In result determination step, introduce colorimetric indicator operation, judge whether each porocyte infects virus by colour-change.The method can only measure cell relative number and relative activity, can not measure cell absolute number, and operation is comparatively loaded down with trivial details.
5) fluorescence quantifying PCR method is by quantitate virus nucleic acid and quantitate virus, and its shortcoming needs loaded down with trivial details method to extract viral DNA, and the method can not distinguish pseudovirion.
The change of cell dia after utilizing virus infected cell is have also appeared to calculate the method for virus titer: after virus infected cell in prior art, breed at cell interior, thus cause the diameter of cell to change, measure virus titer by the intensity of variation of diameter.But, contriver finds in the process verified aforesaid method, in fact and not obvious the diameter change of cells following viral infection, diameter increases usually at about 10-30%, diameter velocity of variation is lower, cause the observed value before and after diameter change too close, not only propose higher requirement to the precision measured, its error measured also will cause measuring viral titre and produce larger departing from relative to theoretical actual value.
Summary of the invention
Technical problem to be solved by this invention is that to measure the method complicated operation of virus titer, the accuracy that measure higher to the skill requirement of operator and precision in prior art not high, and then provides a kind of simple to operate, easy handling person judged result and the method for the mensuration virus titer that measuring result accuracy is high, precision is high.
For this reason, the invention provides a kind of method measuring virus titer, comprise the following steps:
(1) get virus stock solution used and several different dilution viral solution, be inoculated in the cell of virus waiting respectively, cultivate 36-60h, obtain the cell suspension of different concns virus infection;
The described cell suspension infecting virus comprises the nothing dilution cells infected suspension adding virus stock solution used, the dilution cells infected suspension adding each dilution viral solution;
Separately get the cell suspension of described virus waiting as negative controls;
(2) respectively measuring process 1) described in infect the side scatter (S) of cell and the cell density (C) of maintenance intact cell form in the cell suspension of virus and negative controls;
(3) virus titer is calculated according to following step:
A) by formula:
calculate cell infection rate P
m,n, m is viral dilution numbering, and n is the number of replication numbering of same viral dilution cells infected; Wherein, described S
m, nfor the mean value of the cell side scatter of No. m dilution viral n-th group of cells infected suspension; Described
for the mean value of the side scatter of cell in negative controls; Described
for the mean value without the side scatter of cell in dilution cells infected suspension;
B) by formula: N
m,n=C
m,n× P
m,n, calculation procedure a) middle cell infection rate meets 20%<P
m,nthe cells infected density N of the described dilution cells infected suspension of <80%
m,n;
C) by formula: T
m,n=N
m,n/ D
m,n, calculation procedure b) in the virus titer T of each described dilution cells infected suspension
m,n, get the virus titer that median is virus stock solution used; Wherein, described D
m,nfor the extent of dilution of described dilution cells infected suspension.
Further, described step 1) in, before virus inoculation, by the virocyte described waiting of logarithmic phase with 0.6 × 10
6to 1.0 × 10
6the cell density of individual/ml is taped against in Tissue Culture Plate or joins in Suspension Culture Flask, cultivates at 27 DEG C-28 DEG C.
The cell survival rate of described virus waiting is greater than 95%.
Described step 1) in, the extent of dilution of described viral solution is that described dilution cells infected suspension cell infection rate being greater than 80% carries out the extent of dilution of 10 times of dilutions, cell infection rate carries out the extent of dilution half-and-half diluted of 6-8 time lower than the described dilution cells infected suspension of 80%.Described half-and-half dilution, such as first time half-and-half dilution for by solution dilution to be diluted for 1ml to 2ml, second time half-and-half dilution for continuing by solution dilution to 4ml, third time half-and-half dilution for continuing solution dilution to 8ml ..., by that analogy.Certainly, those skilled in the art know, and the extent of dilution got is more, measurement result be more tending towards its theoretic actual value, those skilled in the art can select dilution number according to practical situation.
Step 1) in described virus stock solution used or described viral solution be inoculated in respectively poison cell waiting operation be specially: get isopyknic each described viral solution or described virus stock solution used, inoculum size with 50% is inoculated in the nutrient solution of virocyte waiting, makes virus be 1:1 with the volume ratio of cell.It should be noted that, in the process due to above-mentioned virus inoculation, be equivalent to described viral solution or described virus stock solution used once to dilute again, therefore, described step c) in represent the dilution D of described dilution cells infected suspension
m,nfor the extent of dilution of viral solution described before virus inoculation or described virus stock solution used is multiplied by 50%.
Described step 2) in, adopt Flow Cytometry to measure described side scatter (S), adopt streaming technology or cytometry to measure cell density (C).
Described virus is the one in baculovirus, adenovirus, retrovirus, slow virus.
Described virocyte waiting is insect cell or mammalian cell.
In the present invention, described side scatter (side scatter, SSC) refers to the scattered light signal in 90 ° directions orthogonal with laser beam.Described extent of dilution refers to that solution is by the degree watered down, i.e. the inverse of extension rate, and such as, the solute of 1ml is diluted to 10 times of volume 10ml, then extent of dilution is 1:10, namely 0.1.
Technique scheme of the present invention has the following advantages compared to existing technology:
(1) method of mensuration virus titer of the present invention, utilizes the titre of the change detection virus of the side scatter of virus infected cell.After virocyte cells infected, at cellular proliferative, and then cause intracellular granular number to increase, nucleus also can increase simultaneously, and side scatter increases.Empirical tests, infect the velocity of variation of the side scatter of the cell of virus between 40%-100%, usually more than 60%, change very obvious, by measuring the Δ SSC that different extent of dilution virus infected cell causes, can virus titer be measured through series of computation, avoiding in prior art and measuring the problem that virus titer ubiquitous judged result subjectivity is excessively strong, measurement result is not accurate enough, can objective record data, its measurement result has superior accuracy and precision.
(2) method of mensuration virus titer of the present invention, only need after cell connects poison, measure the side scatter of each cell suspension, just can measure the titre of virus, without the need to the operation such as antibody labeling, staining technique, not only simplify operation, and cost of determination reduces greatly, can realize applying widely.
(3) the present invention measures in the method for virus titer, the cultivation of the 36-60h carried out after the longest required time is only and connects poison, all the other step required times are all shorter, relative to the detection time of at least 5-8 needed for the method such as plaque ethods, Endpoint Dilution Method days, greatly shorten, measurement result can be obtained faster.
Accompanying drawing explanation
In order to make content of the present invention be more likely to be clearly understood, below in conjunction with accompanying drawing, the present invention is further detailed explanation, wherein,
Fig. 1 is the side scatter change schematic diagram of virus infected cell in embodiment 1-5;
Fig. 2 is the Δ SSC velocity of variation comparison diagram in embodiment 1-5 after cell infection virus;
Fig. 3 be in embodiment 1-5 cell infection rate with the change comparison diagram of viral dilution;
Fig. 4 is the measurement result comparison diagram of the inventive method and plaque assay;
Embodiment
Embodiment 1
(1) material
The viral A used in the present embodiment is Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus (AcMNPV) wild-type virus, and construction process is shown in the Bac-to-Bac BaculovirusExpression System. of Invitrogen company; The poison cell waiting used is insect Sf 9 cells, buys in China typical culture collection center, numbering GDC008; Substratum is Sf-900II SFM, adds 10% foetal calf serum (GIBCO, Invitrogen, Carlsbad, CA, USA).Certainly, effect between the product of each commercially available model of above-mentioned materials indifference.
(2) method
The method of the mensuration virus titer of the present embodiment, concrete operations comprise the following steps:
1) by the Insect cells Sf9 of logarithmic phase with 0.8 × 10
6the cell density of individual/ml is taped against in 24 porocyte culture plates, and the volume of the nutrient solution of every Kong Zhonghan poison cell waiting is 250 μ l, cultivates at 27 DEG C, stand-by;
Get the viral A stoste of 250 μ l respectively, the extent of dilution of 250 μ l is 1 × 10
-1, 5 × 10
-2, 2.5 × 10
-2, 1.25 × 10
-2, 6.25 × 10
-3, 3.125 × 10
-3, 1.5625 × 10
-3, 7.8125 × 10
-4, 3.9062 × 10
-4, 1.9531 × 10
-4viral solution, and the cell culture medium of 250 μ l, add in above-mentioned ready described 24 porocyte culture plates, each dilution viral solution does two and repeats multiple hole, and now each hole inner cell density is 0.4 × 10
6individual/ml, cultivates 48h at 27 DEG C, obtains the cell suspension and the negative controls that infect virus;
2) adopt flow cytometer respectively measuring process 1) described in infect the cell suspension of virus and the side scatter S of negative controls and keep the cell density C of intact cell form, the observed value in two multiple holes is designated as R1, R2 respectively;
3) virus titer is calculated according to following step:
A) by formula:
calculate cell infection rate P
m,n, m is viral dilution numbering, and n is the number of replication numbering of same viral dilution cells infected; Wherein, described S
m,nfor the mean value of the cell side scatter of m extent of dilution virus n-th group of cells infected suspension; Described
for the mean value of the side scatter of cell in negative controls; Described
for the mean value without the side scatter of cell in dilution cells infected suspension; Infection rate is designated as 1 higher than 0.99, and infection rate is designated as 0 lower than 0.01;
B) by formula: N
m,n=C
m,n× P
m,n, calculation procedure is middle cell infection rate 20%<P a)
m,nthe cells infected density N in each hole during <80%
m,n;
C) T
m,n=N
m,n/ D
m,n, calculation procedure b) in the virus titer T in each hole (bottle)
m,n, D
m,nfor the extent of dilution before this hole virus inoculation is multiplied by virus inoculation amount 50%.To a series of T of gained
m,nget the virus titer (IU/ml) that median is virus stock solution used.
(3) result
Its measurement result is as shown in the table:
Table 1 viral A titer determination result
Its measuring principle as shown in Figure 1, poison cell waiting is before virus inoculation, granule number is less, after described virus infection poison cell waiting, at cellular proliferative, intracellular granular number is increased, the side scatter infected before and after virus is changed, i.e. Δ SSC, through measuring the side scatter mean value of the cell suspension that different dilution viral solution infects, can determine virus titer.
Embodiment 2
(1) material
The viral B used in the present embodiment is for expressing the Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus (AcMNPV) of EGFP, and construction process is shown in the Bac-to-Bac BaculovirusExpression System of Invitrogen company; The poison cell waiting used is insect Sf 9 cells, buys in China typical culture collection center, numbering GDC008; Substratum is Sf-900II SFM, adds 10% foetal calf serum (GIBCO, Invitrogen, Carlsbad, CA, USA).Certainly, effect between the product of each commercially available model of above-mentioned materials indifference.
(2) method
The method of the mensuration virus titer of the present embodiment, concrete operations comprise the following steps:
1) by the Insect cells Sf9 of logarithmic phase with 0.8 × 10
6the cell density of individual/ml is taped against in 24 porocyte culture plates, and the volume of the nutrient solution of every Kong Zhonghan poison cell waiting is 250 μ l, cultivates at 27 DEG C, stand-by;
Get the viral B stoste of 250 μ l respectively, the extent of dilution of 250 μ l is 1 × 10
-1, 5 × 10
-2, 2.5 × 10
-2, 1.25 × 10
-2, 6.25 × 10
-3, 3.125 × 10
-3, 1.5625 × 10
-3, 7.8125 × 10
-4, 3.9062 × 10
-4, 1.9531 × 10
-4viral solution, and the cell culture medium of 250 μ l, add in above-mentioned ready described 24 porocyte culture plates, each dilution viral solution does two and repeats multiple hole, and now each hole inner cell density is 0.4 × 10
6individual/ml, cultivates 48h at 27 DEG C, obtains the cell suspension and the negative controls that infect virus;
2) adopt flow cytometer respectively measuring process 1) described in infect the cell suspension of virus and the side scatter S of negative controls and keep the cell density C of intact cell form, the observed value in two multiple holes is designated as R1, R2 respectively;
3) virus titer is calculated according to following step:
A) by formula:
calculate cell infection rate P
m,n, m is viral dilution numbering, and n is the number of replication numbering of same viral dilution cells infected; Wherein, described S
m,nfor the mean value of the cell side scatter of m extent of dilution virus n-th group of cells infected suspension; Described
for the mean value of the side scatter of cell in negative controls; Described
for the mean value without the side scatter of cell in dilution cells infected suspension; Infection rate is designated as 1 higher than 0.99, and infection rate is designated as 0 lower than 0.01;
B) by formula: N
m,n=C
m,n× P
m,n, calculation procedure is middle cell infection rate 20%<P a)
m,nthe cells infected density N in each hole during <80%
m,n;
C) T
m,n=N
m,n/ D
m,n, calculation procedure b) in the virus titer T in each hole (bottle)
m,n, D
m,nfor the extent of dilution before this hole virus inoculation is multiplied by virus inoculation amount 50%.To a series of T of gained
m,nget the virus titer (IU/ml) that median is virus stock solution used.
(3) result
Its measurement result is as shown in the table:
Table 2 viral B titer determination result
Embodiment 3
(1) material
The viral C used in the present embodiment is for expressing the Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus (AcMNPV) of AAV rep albumen, and construction process is shown in the Bac-to-BacBaculovirus Expression System of Invitrogen company; The poison cell waiting used is insect Sf 9 cells, buys in China typical culture collection center, numbering GDC008; Substratum is Sf-900II SFM, adds 10% foetal calf serum (GIBCO, Invitrogen, Carlsbad, CA, USA).Certainly, effect between the product of each commercially available model of above-mentioned materials indifference.
(2) method
The method of the mensuration virus titer of the present embodiment, concrete operations comprise the following steps:
1) by the Insect cells Sf9 of logarithmic phase with 0.8 × 10
6the cell density of individual/ml is taped against in 24 porocyte culture plates, and the volume of the nutrient solution of every Kong Zhonghan poison cell waiting is 250 μ l, cultivates at 27 DEG C, stand-by;
Get the viral C stoste of 250 μ l respectively, the extent of dilution of 250 μ l is 1 × 10
-1, 5 × 10
-2, 2.5 × 10
-2, 1.25 × 10
-2, 6.25 × 10
-3, 3.125 × 10
-3, 1.5625 × 10
-3, 7.8125 × 10
-4, 3.9062 × 10
-4, 1.9531 × 10
-4viral solution, and the cell culture medium of 250 μ l, add in above-mentioned ready described 24 porocyte culture plates, each dilution viral solution does two and repeats multiple hole, and now each hole inner cell density is 0.4 × 10
6individual/ml, cultivates 48h at 27 DEG C, obtains the cell suspension and the negative controls that infect virus;
2) adopt flow cytometer respectively measuring process 1) described in infect the cell suspension of virus and the side scatter S of negative controls and keep the cell density C of intact cell form, the observed value in two multiple holes is designated as R1, R2 respectively;
3) virus titer is calculated according to following step:
A) by formula:
calculate cell infection rate P
m,n, m is viral dilution numbering, and n is the number of replication numbering of same viral dilution cells infected; Wherein, described S
m,nfor the mean value of the cell side scatter of m extent of dilution virus n-th group of cells infected suspension; Described
for the mean value of the side scatter of cell in negative controls; Described
for the mean value without the side scatter of cell in dilution cells infected suspension; Infection rate is designated as 1 higher than 0.99, and infection rate is designated as 0 lower than 0.01;
B) by formula: N
m,n=C
m,n× P
m,n, calculation procedure is middle cell infection rate 20%<P a)
m,nthe cells infected density N in each hole during <80%
m,n;
C) T
m,n=N
m,n/ D
m,n, calculation procedure b) in the virus titer T in each hole (bottle)
m,n, D
m,nfor the extent of dilution before this hole virus inoculation is multiplied by virus inoculation amount 50%.To a series of T of gained
m,nget the virus titer (IU/ml) that median is virus stock solution used.
(3) result
Its measurement result is as shown in the table:
Table 3 viral C titer determination result
Embodiment 4
(1) material
The viral D used in the present embodiment is for expressing the Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus (AcMNPV) of AAVcap albumen, and construction process is shown in the Bac-to-BacBaculovirus Expression System of Invitrogen company; The poison cell waiting used is insect Sf 9 cells, buys in China typical culture collection center, numbering GDC008; Substratum is Sf-900II SFM, adds 10% foetal calf serum (GIBCO, Invitrogen, Carlsbad, CA, USA).Certainly, effect between the product of each commercially available model of above-mentioned materials indifference.
(2) method
The method of the mensuration virus titer of the present embodiment, concrete operations comprise the following steps:
1) by the Insect cells Sf9 of logarithmic phase with 0.6 × 10
6the cell density of individual/ml is taped against in 24 porocyte culture plates, and the volume of the nutrient solution of every Kong Zhonghan poison cell waiting is 250 μ l, cultivates at 27 DEG C, stand-by;
Get the viral C stoste of 250 μ l respectively, the extent of dilution of 250 μ l is 1 × 10
-1, 5 × 10
-2, 2.5 × 10
-2, 1.25 × 10
-2, 6.25 × 10
-3, 3.125 × 10
-3, 1.5625 × 10
-3, 7.8125 × 10
-4, 3.9062 × 10
-4, 1.9531 × 10
-4viral solution, and the cell culture medium of 250 μ l, add in above-mentioned ready described 24 porocyte culture plates, each dilution viral solution does two and repeats multiple hole, and now each hole inner cell density is 0.3 × 10
6individual/ml, cultivates 48h at 27 DEG C, obtains the cell suspension and the negative controls that infect virus;
2) adopt flow cytometer respectively measuring process 1) described in infect the cell suspension of virus and the side scatter S of negative controls and keep the cell density C of intact cell form, the observed value in two multiple holes is designated as R1, R2 respectively;
3) virus titer is calculated according to following step:
A) by formula:
calculate cell infection rate P
m,n, m is viral dilution numbering, and n is the number of replication numbering of same viral dilution cells infected; Wherein, described S
m,nfor the mean value of the cell side scatter of m extent of dilution virus n-th group of cells infected suspension; Described
for the mean value of the side scatter of cell in negative controls; Described
for the mean value without the side scatter of cell in dilution cells infected suspension; Infection rate is designated as 1 higher than 0.99, and infection rate is designated as 0 lower than 0.01;
B) by formula: N
m,n=C
m,n× P
m,n, calculation procedure is middle cell infection rate 20%<P a)
m,nthe cells infected density N in each hole during <80%
m,n;
C) T
m,n=N
m,n/ D
m,n, calculation procedure b) in the virus titer T in each hole (bottle)
m,n, D
m,nfor the extent of dilution before this hole virus inoculation is multiplied by virus inoculation amount 50%.To a series of T of gained
m,nget the virus titer (IU/ml) that median is virus stock solution used.
(3) result
Its measurement result is as shown in the table:
Table 4 viral D titer determination result
Embodiment 5
(1) material
The viral E used in the present embodiment is for expressing the Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus (AcMNPV) of coagulagen coaglogen, and construction process is shown in the Bac-to-BacBaculovirus Expression System of Invitrogen company; The poison cell waiting used is insect Sf 9 cells, buys in China typical culture collection center, numbering GDC008; Substratum is Sf-900II SFM, adds 10% foetal calf serum (GIBCO, Invitrogen, Carlsbad, CA, USA).Certainly, effect between the product of each commercially available model of above-mentioned materials indifference.
(2) method
The method of the mensuration virus titer of the present embodiment, concrete operations comprise the following steps:
1) by the Insect cells Sf9 of logarithmic phase with 1.0 × 10
6the cell density of individual/ml is taped against in 24 porocyte culture plates, and the volume of the nutrient solution of every Kong Zhonghan poison cell waiting is 250 μ l, cultivates at 27 DEG C, stand-by;
Get the viral C stoste of 250 μ l respectively, the extent of dilution of 250 μ l is 1 × 10
-1, 5 × 10
-2, 2.5 × 10
-2, 1.25 × 10
-2, 6.25 × 10
-3, 3.125 × 10
-3, 1.5625 × 10
-3, 7.8125 × 10
-4, 3.9062 × 10
-4, 1.9531 × 10
-4viral solution, and the cell culture medium of 250 μ l, add in above-mentioned ready described 24 porocyte culture plates, each dilution viral solution does two and repeats multiple hole, and now each hole inner cell density is 0.5 × 10
6individual/ml, cultivates 36h at 27 DEG C, obtains the cell suspension and the negative controls that infect virus;
2) adopt flow cytometer respectively measuring process 1) described in infect the cell suspension of virus and the side scatter S of negative controls and keep the cell density C of intact cell form, the observed value in two multiple holes is designated as R1, R2 respectively;
3) virus titer is calculated according to following step:
A) by formula:
calculate cell infection rate P
m,n, m is viral dilution numbering, and n is the number of replication numbering of same viral dilution cells infected; Wherein, described S
m,nfor the mean value of the cell side scatter of m extent of dilution virus n-th group of cells infected suspension; Described
for the mean value of the side scatter of cell in negative controls; Described
for the mean value without the side scatter of cell in dilution cells infected suspension; Infection rate is designated as 1 higher than 0.99, and infection rate is designated as 0 lower than 0.01;
B) by formula: N
m,n=C
m,n× P
m,n, calculation procedure is middle cell infection rate 20%<P a)
m,nthe cells infected density N in each hole during <80%
m,n;
C) T
m,n=N
m,n/ D
m,n, calculation procedure b) in the virus titer T in each hole (bottle)
m,n, D
m,nfor the extent of dilution before this hole virus inoculation is multiplied by virus inoculation amount 50%.To a series of T of gained
m,nget the virus titer (IU/ml) that median is virus stock solution used.
(3) result
Its measurement result is as shown in the table:
Table 5 viral E titer determination result
Effect experimental examples
For verifying the method for mensuration virus titer of the present invention, adopt plaque ethods to measure the virus identical with embodiment 1-5 and virocyte waiting respectively, carry out the mensuration of virus titer, and contrast in method of the present invention, concrete operations are as follows:
The cell density of adjustment Sf9 is 5 × 10
5cell/ml, adds in six orifice plates with the amount of every hole 2ml, is placed in the cell culture incubator adherent culture 1h of 27 DEG C.With Sf-900II SFM substratum virus stock solution used 10 times dilution: 10
-1-10
-8.Discard the supernatant liquor in six orifice plates, add 10
-5-10
-8dilution viral solution, each extent of dilution does 2 multiple holes.The cell culture incubator being placed in 27 DEG C hatches 1h.Discard viral supernatants, every hole adds the top-layer agar (1 × Sf-900II SFM, 10%FBS, 1% low melting-point agarose) that 2ml configures fast.The cell culture incubator being placed in 27 DEG C is cultivated after 4 days, top-layer agar adds 1ml neutral-red agar (1 × Sf-900II SFM, 62.5 μ g/ml toluylene reds, 1% low melting-point agarose), put into 27 DEG C of incubators again to continue to cultivate, until there is obvious plaque, when the number of plaque no longer changes, count the plaque number in every hole, plaque number, in the hole of 5-50 scope, calculates virus titer according to the following equation: the every hole inoculation in virus titer (pfu/ml)=1/ extension rate × plaque number × 1/ volume.
Its measurement result is as follows:
Table 6 virus titer measurement result contrasts
Be illustrated in figure 4 the comparison diagram that plaque assay and method of the present invention measure virus titer result, from upper table and Fig. 4, the result that the virus titer of method mensuration of the present invention and plaque ethods measure is close, and standard variance is less, namely measurement result divides spread of distribution little, accuracy and precision better.
Obviously, above-described embodiment is only for clearly example being described, and the restriction not to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.And thus the apparent change of extending out or variation be still among the protection domain of the invention.
Claims (8)
1. measure a method for virus titer, it is characterized in that, comprise the following steps:
(1) get virus stock solution used and several different dilution viral solution, be inoculated in the cell of virus waiting respectively, cultivate 36-60h, obtain the cell suspension of different concns virus infection;
The described cell suspension infecting virus comprises the nothing dilution cells infected suspension adding virus stock solution used, the dilution cells infected suspension adding each dilution viral solution;
Separately get the cell suspension of described virus waiting as negative controls;
(2) respectively measuring process 1) described in infect the side scatter (S) of cell and the cell density (C) of maintenance intact cell form in the cell suspension of virus and negative controls;
(3) virus titer is calculated according to following step:
A) by formula:
calculate cell infection rate P
m,n, m is viral dilution numbering, and n is the number of replication numbering of same viral dilution cells infected; Wherein, described S
m,nfor the mean value of the cell side scatter of No. m dilution viral n-th group of cells infected suspension; Described
for the mean value of the side scatter of cell in negative controls; Described
for the mean value without the side scatter of cell in dilution cells infected suspension;
B) by formula: N
m,n=C
m,n× P
m,n, calculation procedure a) middle cell infection rate meets 20%<P
m,nthe cells infected density N of the described dilution cells infected suspension of <80%
m,n;
C) by formula: T
m,n=N
m,n/ D
m,n, calculation procedure b) in the virus titer T of each described dilution cells infected suspension
m,n, get the virus titer that median is virus stock solution used; Wherein, described D
m,nfor the extent of dilution of described dilution cells infected suspension.
2. the method for mensuration virus titer according to claim 1, is characterized in that:
Described step 1) in, before virus inoculation, by the virocyte described waiting of logarithmic phase with 0.6 × 10
6to 1.0 × 10
6the cell density of individual/ml is taped against in Tissue Culture Plate or joins in Suspension Culture Flask, cultivates at 27 DEG C-28 DEG C.
3. the method for mensuration virus titer according to claim 2, is characterized in that: the cell survival rate of described virus waiting is greater than 95%.
4. according to the method for described mensuration virus titer arbitrary in claim 1-3, it is characterized in that: described step 1) in, the extent of dilution of described viral solution is that described dilution cells infected suspension cell infection rate being greater than 80% carries out the extent of dilution of 10 times of dilutions, cell infection rate carries out the extent of dilution half-and-half diluted of 6-8 time lower than the described dilution cells infected suspension of 80%.
5. according to the method for the arbitrary described mensuration virus titer of claim 1-4, it is characterized in that: step 1) in described virus stock solution used or described viral solution be inoculated in respectively virocyte waiting operation be specially: get isopyknic each described viral solution or described virus stock solution used, inoculum size with 50% is inoculated in the nutrient solution of virocyte waiting, makes virus be 1:1 with the volume ratio of cell.
6. according to the method for the arbitrary described mensuration virus titer of claim 1-5, it is characterized in that: described step 2) in, adopt Flow Cytometry to measure described side scatter (S), adopt streaming technology or cytometry to measure cell density (C).
7., according to the method for the arbitrary described mensuration virus titer of claim 1-6, it is characterized in that: described virus is the one in baculovirus, adenovirus, retrovirus, slow virus.
8., according to the method for the arbitrary described mensuration virus titer of claim 1-7, it is characterized in that: described virocyte waiting is insect cell or mammalian cell.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106048090A (en) * | 2016-07-05 | 2016-10-26 | 华中农业大学 | Method of quickly measuring titer of ascovirus |
| CN111366525A (en) * | 2020-03-12 | 2020-07-03 | 西安交通大学 | Method for rapidly detecting SARS-CoV-2 virus infection in isolated blood sample and application thereof |
| CN115181816A (en) * | 2022-08-10 | 2022-10-14 | 杭州纽创生物检测有限公司 | Method for detecting virus titer in ultralow virus titer system and application thereof |
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| US6248514B1 (en) * | 1996-07-09 | 2001-06-19 | Canji, Inc. | Methods for measuring viral infectivity |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106048090A (en) * | 2016-07-05 | 2016-10-26 | 华中农业大学 | Method of quickly measuring titer of ascovirus |
| CN111366525A (en) * | 2020-03-12 | 2020-07-03 | 西安交通大学 | Method for rapidly detecting SARS-CoV-2 virus infection in isolated blood sample and application thereof |
| CN111366525B (en) * | 2020-03-12 | 2021-08-13 | 西安交通大学 | Method for rapidly detecting SARS-CoV-2 virus infection in isolated blood sample and application thereof |
| CN115181816A (en) * | 2022-08-10 | 2022-10-14 | 杭州纽创生物检测有限公司 | Method for detecting virus titer in ultralow virus titer system and application thereof |
| CN115181816B (en) * | 2022-08-10 | 2023-01-06 | 杭州纽创生物检测有限公司 | Detection method for virus titer in ultra-low virus titer system and application thereof |
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